Scanning optical microscope

ABSTRACT

A scanning optical microscope using a wavefront converting element suffers minimum off-axis performance degradation and allows the wavefront converting element to be controlled by a simple method. Further, a pupil relay optical system is simple in arrangement or unnecessary. A laser scanning microscope includes a laser oscillator  6  and a wavefront converting element  5  for applying a desired wavefront conversion to a laser beam  15  emitted from the laser oscillator  6 . An objective  7  collects a wavefront-converted approximately parallel laser beam  17  emerging from the wavefront converting element  5  onto a sample  9 . A detector  29  detects signal light emitted from the sample  9 . An actuator 8 scans the objective  7  along a direction perpendicular to the optical axis.

[0001] This application claims benefit of Japanese Application No.2000-394934 filed in Japan on Dec. 26, 2000, the contents of which areincorporated by this reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention The present invention relates toscanning optical microscopes and, more particularly, to a laser scanningmicroscope (LSM) that performs focal point movement along the directionof the optical axis by using a wavefront converting element.

[0003] 2. Discussion of Related Art

[0004] It has heretofore been necessary in order to obtain athree-dimensional image of a specimen with an LSM, for example, tocapture optical images of successive planes inside the specimen bymechanically moving either the specimen or the objective along thedirection of the optical axis. With this method, however, it isdifficult to realize positional control with high accuracy and highreproducibility because the method needs mechanical drive. In a casewhere the specimen is moved, high-speed scanning cannot be effected whenthe specimen is large in size.

[0005] In observation of a biological specimen, if the objectiveis-scanned in the state of being in direct contact with the specimen orimmersed in a culture solution of the specimen, vibrations of theobjective adversely affect the specimen under observation.

[0006] To solve the above-described problems, Japanese PatentApplication Unexamined Publication (KOKAI) No. Hei 11-101942 disclosesan adaptive optical apparatus. The apparatus is a microscope having anoptical element (wavefront converting element) capable of changingpower. The arrangement of the microscope is shown in FIGS. 27 and 28. Inthis prior art, a wavefront converting element is inserted in either orboth of a viewing optical path and an illuminating optical path tochange the focal length of the optical system and to correct aberrationdue to the change of the focal length by using the wavefront convertingelement. With this arrangement, it is possible not only to form and movea focal point in the object space without changing the distance betweenthe objective and the specimen but also to correct aberration.

[0007] In the above-described prior art, it is preferable to place thewavefront converting element in the pupil plane of the objective or at aposition conjugate to the pupil plane from the viewpoint of allowing thewavefront converting element to effectively perform its functions ofmoving the focal point in the object space and making aberrationcorrection. If the wavefront converting element is not conjugate to thepupil plane, illuminating light or image-forming light will pass atdifferent positions on the wavefront converting element according to theheight of the object detected by the objective. To perform focal pointmovement or aberration correction, the wavefront shape has to be changedaccording to the object height. If the wavefront shape cannot properlybe changed, image quality is likely to degrade considerably in an areawhere the object height is high.

[0008] If the wavefront converting element is changed into an optimumshape in accordance with a change in the object height, even if thewavefront converting element is not conjugate to the pupil plane, it ispossible to avoid image quality degradation in an area where the objectheight is high. To realize this, however, the wavefront convertingelement needs to be controlled at high speed so as to provide an optimumrotationally asymmetric configuration. This is extremely difficult.

[0009] For the reasons stated above, it is desirable that the wavefrontconverting element should be placed at a position conjugate to thepupil. This is, however, difficult to implement because of the followingproblems.

[0010] A variety of objectives are used in microscopic observation, andthe pupil position differs for each objective. Therefore, when aplurality of objectives are switched from one to another to performobservation, it is difficult to keep the pupils of the objectives inconjugate relation to the wavefront converting element at all times.

[0011] Further, the wavefront converting element needs to be placed inconjugate relation to the position of a laser scanning member and alsoto the position of the objective pupil. Accordingly, at least two pupilrelay optical systems are required. Therefore, the apparatus becomeslarge in size and complicated unfavorably.

[0012] Further, in the above-described prior art, a reflection typewavefront converting element is incorporated in the illuminating opticalpath or/and the light-detecting optical path. Therefore, the prior artuses beam splitters as shown in FIGS. 27 and 28. Accordingly, when anon-polarized laser is used as a light source, together with anon-polarization type beam splitter, the amount of light is reduced to¼every time the laser beam travels via the wavefront converting element.

[0013] More specifically, the amount of light is reduced to ¼in theprocess of illumination and also reduced to ¼in the process ofdetection. That is, the amount of light is reduced to {fraction(1/16)}in total. If a linearly polarized laser is used as a lightsource, together with a polarization beam splitter and a quarter-waveplate, the loss of light in the process of illumination can beprevented. However, in observation of fluorescence in a non-polarizedstate, the amount of light is reduced to ½in the process of(fluorescence) detection.

[0014] Further, even when a polarization beam splitter and aquarter-wave plate are used as stated above, it is not always possibleto use a linearly polarized laser as a light source. If a non-polarizedlaser is used to observe fluorescence, the amount of light is reduced to½in the process of illumination and also reduced to ½in the process ofdetection. That is, the amount of light is reduced to ¼in total.

SUMMARY OF THE INVENTION

[0015] The present invention was made to solve the above- describedproblems associated with the prior art. Objects of the present inventionare as follows. A first object of the present invention is to provide ascanning optical microscope, e.g. a laser scanning microscope (LSM),using a wavefront converting element, wherein even when the object pupiland the wavefront converting element are not placed in conjugaterelation to each other, off-axis performance degradation is minimized,and wherein the wavefront converting element can be controlled by anextremely simple method, and a pupil relay optical system is simple inarrangement or unnecessary. A second object of the present invention isto provide an LSM using a wavefront converting element, in which theloss of light can be prevented even when the wavefront convertingelement is of the reflection type.

[0016] To attain the above-described objects, the present inventionprovides a first scanning optical microscope including a light sourceand a wavefront converting element for applying a desired wavefrontconversion to illuminating light emitted from the light source. Anobjective collects wavefront-converted illuminating light emerging fromthe wavefront converting element onto a sample. A detector detectssignal light emitted from the sample. An actuator scans the objectivealong a direction perpendicular to the optical axis.

[0017] It is desirable that illuminating light emerging from thewavefront converting element should be an approximately parallel beam.

[0018] In addition, the present invention provides a second scanningoptical microscope wherein when the above- described actuator scans onesection of the sample perpendicular to the optical axis with theobjective, the wavefront converting element applies a constant wavefrontconversion to the illuminating light.

[0019] In addition, the present invention provides a third scanningoptical microscope having an arrangement similar to that of the first orsecond scanning optical microscope, wherein when the amount of movementof the objective along the direction perpendicular to the optical axis(this will hereinafter be referred to as “scan range”) is denoted by ΔX,the following condition (1) is satisfied:

ΔX≦0.66f_(OR)·λ(ΔX·NA⁴)   (1)

[0020] where:

[0021] f_(OB):the focal length of the objective;

[0022] ΔZ:the amount of focal point movement caused by the wavefrontconverting element;

[0023] λ:the wavelength of the illuminating light;

[0024] NA:the numerical aperture of the objective.

[0025] In addition, the present invention provides a fourth scanningoptical microscope including a light source and an optical elementhaving a positive power for converting illuminating light emitted fromthe light source into a convergent beam. The fourth scanning opticalmicroscope further includes a reflecting mirror with an aperture and areflection type wavefront converting element for applying a desiredwavefront conversion to the illuminating light. An objective collectsthe wavefront-converted illuminating light onto a sample. A detectordetects signal light emitted from the sample.

[0026] In addition, the present invention provides a fifth scanningoptical microscope wherein an optical system including the reflectingmirror with an aperture in the fourth scanning optical microscopesatisfies the following condition (2):

r _(Hmin)/r_(Minc)≦0.5   (2)

[0027] where:

[0028] r_(Hmin):the minimum value of the length from the optical axis tothe reflecting mirror edge;

[0029] r_(Minc):the radius of wavefront-converted illuminating lightincident on the reflecting mirror with an aperture.

[0030] In addition, the present invention provides a sixth scanningoptical microscope including a light source and an optical elementhaving a positive power for converting illuminating light emitted fromthe light source into a convergent beam. A reflecting mirror is placedat a position where the convergent beam is collected. A reflection typewavefront converting element applies a desired wavefront conversion tothe illuminating light. An objective collects the wavefront-convertedilluminating light onto a sample. A detector detects signal lightemitted from the sample.

[0031] In addition, the present invention provides a seventh scanningoptical microscope wherein an optical system including the reflectingmirror in the sixth scanning optical microscope satisfies the followingcondition (3):

r _(Mmin)/r_(Ainc)≦0.5   (3)

[0032] where:

[0033] r_(Mmin):the minimum value of the length from the optical axis tothe reflecting mirror edge;

[0034] r_(Ainc):the radius of wavefront-converted illuminating light atthe position of the reflecting mirror.

[0035] In addition, the present invention provides an eighth scanningoptical microscope including a light source and a reflection typewavefront converting element for applying a desired wavefront conversionto illuminating light emitted from the light source. An objectivecollects wavefront-converted illuminating light onto a sample. The lightsource also serves as a detector for detecting signal light emitted fromthe sample.

[0036] In addition, the present invention provides a ninth scanningoptical microscope including a light source and a reflection typewavefront converting element for applying a desired wavefront conversionto illuminating light emitted from the light source. An objectivecollects wavefront-converted illuminating light emerging from thewavefront converting element onto a sample. A detector detects signallight emitted from the sample. The reflection type wavefront convertingelement is placed in an optical path so as to satisfy the followingcondition (4):

θ_(PR)≦50·NA⁻¹{square root}(λ·ΔZ⁻¹⁾  (4)

[0037] where:

[0038] θ_(PR):the angle (°) of incidence of the principal ray on thewavefront converting element;

[0039] ΔZ:the amount of focal point movement;

[0040] λ:the wavelength of the illuminating light;

[0041] NA:the numerical aperture of the objective.

[0042] In addition, the present invention provides a tenth scanningoptical microscope including a light source and a reflection typewavefront converting element for applying a wavefront conversion toilluminating light emitted from the light source. An objective collectswavefront-converted illuminating light emerging from the wavefrontconverting element onto a sample. A detector detects signal lightemitted from the sample. The reflecting surface of the reflection typewavefront converting element is controllable into an aspherical toricsurface configuration.

[0043] Still other objects and advantages of the invention will in partbe obvious and will in part be apparent from the specification.

[0044] The invention accordingly comprises the features of construction,combinations of elements, and arrangement of parts which will beexemplified in the construction hereinafter set forth, and the scope ofthe invention will be indicated in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0045]FIG. 1 is a diagram showing the arrangement of a laser scanningmicroscope according to the present invention wherein focal pointmovement is made by a wavefront converting element, and XY-scanning isperformed by driving an objective.

[0046] FIGS. 2(a) and 2(b) are diagrams for describing the plus-sidemovement and minus-side movement of a focal point caused by thewavefront converting element.

[0047]FIG. 3 is a diagram for describing XY-scanning with the objective.

[0048]FIG. 4 is a diagram for describing wavefront aberration due to themovement of the focal point.

[0049]FIG. 5 is a diagram for describing wavefront aberration due toscanning with the objective.

[0050]FIG. 6 is a graph showing the results of simulation for obtaininga scan range ΔX [when f_(OB)=3 (mm)] where the Strehl ratio is 0.7 ormore, together with the curves of formula (1).

[0051]FIG. 7 is a graph showing the results of simulation for obtaininga scan range ΔX [when f_(OB)=10 (mm)] where the Strehl ratio is 0.7 ormore, together with the curves of formula (1).

[0052]FIG. 8 is a diagram showing the arrangement of an embodiment of alaser scanning microscope having a reflection type wavefront convertingelement and a reflecting mirror with an aperture.

[0053]FIG. 9 is a diagram showing the arrangement of another embodimentof the laser scanning microscope having a reflection type wavefrontconverting element and a reflecting mirror with an aperture.

[0054]FIG. 10 is a diagram showing the arrangement of an embodiment of alaser scanning microscope having a galvanometer mirror, a reflectiontype wavefront converting element and a reflecting mirror with anaperture.

[0055]FIG. 11 is a diagram for describing the loss of light at thereflecting mirror with an aperture shown in FIG. 10.

[0056] FIGS. 12(a), 12(b) and 12(c) are diagrams each showing the beamdiameter at a reflecting mirror with an aperture and the shape of theaperture as projected.

[0057]FIG. 13 is a diagram showing the intensity distribution of aGaussian beam.

[0058]FIG. 14 is a diagram showing the arrangement of an embodiment of alaser scanning microscope having a reflection type wavefront convertingelement and a reflecting mirror.

[0059]FIG. 15 is a diagram for describing the loss of light at thereflecting mirror shown in FIG. 14.

[0060]FIG. 16 is a diagram showing the beam diameter at a reflectingmirror and the shape of a reflecting surface as projected.

[0061]FIG. 17 is a diagram showing the arrangement of an embodiment of alaser scanning microscope having a reflection type wavefront convertingelement and an optical element serving as both a light-emitting part anda light-receiving part.

[0062]FIG. 18 is a diagram showing the arrangement of an embodiment of alaser scanning microscope having a reflection type wavefront convertingelement and an optical fiber.

[0063]FIG. 19 is a diagram showing the arrangement of an embodiment of alaser scanning microscope in which a light beam is incident obliquely ona reflection type wavefront converting element.

[0064]FIG. 20 is a diagram showing the arrangement of an embodiment of alaser scanning microscope in which a light beam is incident obliquely ona reflection type wavefront converting element through a collimationlens.

[0065]FIG. 21 is a graph showing the results of simulation for obtainingan obliquely incident angle Δθ_(PR) [when ΔZ=0.05 (mm)] at which theStrehl ratio is 0.7 or more, together with the curves of formula (4).

[0066]FIG. 22 is a graph showing the results of simulation for obtainingan obliquely incident angle Δθ_(PR) [when ΔZ=0.02 (mm)] at which theStrehl ratio is 0.7 or more, together with the curves of formula (4).

[0067]FIG. 23 is a diagram showing the arrangement of an embodiment of alaser scanning microscope using a reflection type wavefront convertingelement having an aspherical toric surface.

[0068]FIG. 24 is a diagram showing a simulation model of an objectivescanning microscope having a reflection type wavefront convertingelement with an aspherical toric surface.

[0069]FIG. 25 is a contour map showing an optimized reflecting surfaceconfiguration when a free-form surface type reflection wavefrontconverting element is used.

[0070]FIG. 26 is a contour map showing a reflecting surfaceconfiguration obtained by subtracting an optimized toric surface fromthe optimized free-form surface shown in FIG. 25.

[0071]FIG. 27 is a diagram showing the arrangement of a conventionalmicroscope in which a beam splitter is used for optical path splitting.

[0072]FIG. 28 is a diagram showing the arrangement of a conventionaltwo-photon microscope in which beam splitters are used for optical pathsplitting.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0073] The arrangement and operation of the scanning optical microscopesaccording to the present invention will be described below specificallywith reference to the accompanying drawings. It should be noted that thesame elements repeatedly employed in the drawings, which are used forthe following description, are denoted by the same reference numerals,and a redundant description thereof is not given. Further, the presentinvention will be described as a laser scanning microscope (LSM) using alaser oscillator as a light source.

[0074] The basic arrangement of the first scanning optical microscopeaccording to the present invention, together with wavefront aberrationdue to the movement of the object point during Z-scan (i.e. scan alongthe optical axis direction) and a variation of the arrangement, will bedescribed below with reference to FIGS. 1 to 4.

[0075] An LSM can be realized by an arrangement as shown in FIG. 1. InFIG. 1, a laser light source 6 emits illuminating light 2. Theilluminating light 2 is converted into a plane wave through acollimation lens 4. The plane wave passes through a beam splitter 21 andenters a wavefront converting element 5 as pre-correction illuminatinglight 15. In the wavefront converting element 5, the pre-correctionilluminating light 15 is subjected to a predetermined wavefrontconversion (described later) and exits therefrom as post-correctionilluminating light 17, which is then converted into a spherical wavethrough an objective 7 to illuminate a point on a sample 9. Reflectedlight from the sample 9 is collected through the objective 7 and entersthe wavefront converting element 5 as pre-correction viewing light 18.In the wavefront converting element 5, the pre-correction viewing light18 is subjected to a predetermined wavefront conversion and exitstherefrom as post-correction viewing light 16, which is a plane wave.The post-correction viewing light 16 is reflected by the beam splitter21 and collected through a convex lens 28 to enter a photo-detector 29as viewing light 3.

[0076] The objective 7 is of the infinity corrected type, in whichaberration is minimized when the object plane is coincident with theobject-side focal point F thereof. An objective actuator 8 scans theobjective 7 along the XY-directions.

[0077] The wavefront converting element 5 is capable of converting thewavefront of illuminating light. Therefore, as shown in FIG. 2(a), thepost-correction illuminating light 17 can be formed into a divergentbeam wavefront 13 so that the position where the illuminating light iscollected shifts to a plus-side shifted focal point 11, which is moreaway from the objective 7 than the object-side focal point F.Conversely, as shown in FIG. 2(b), the post-correction illuminatinglight 17 can also be formed into a convergent beam wavefront 14 so thatthe position where the illuminating light is collected shifts to aminus-side shifted focal point 12, which is closer to the objective 7than the object-side focal point F. That is, the position where theilluminating light is collected can be moved without moving theobjective 7 or the sample 9.

[0078] Further, because the objective 7 is scanned along theXY-directions by the objective actuator 8, the object-side focal point Fcan be moved along the XY-directions, as shown in FIG. 3. In the figure,OA₁ denotes the optical axis of the wavefront converting element 5, andOA₂ denotes the optical axis of the objective 7. Scan range ΔX is thedistance between OA₁ and OA_(2.)

[0079] In other words, this system functions as a laser scanningmicroscope in which a position where a laser beam is collected isspatially moved to illuminate the sample 9, and light from the sample 9is detected as viewing light.

[0080] Let us explain the operation of the wavefront converting element5 to move the focal position as shown in FIGS. 2(a) and 2(b).

[0081] Because the objective 7 is of the infinity corrected type, whenthe illuminating light source is at a finite distance, the focalposition is displaced from the object-side focal point F. In addition,spherical aberration increases. Accordingly, in order to effectfavorable image formation at the plus-side shifted focal point 11 andthe minus-side shifted focal point 12, it is desirable that thedivergent beam wavefront 13 and the convergent beam wavefront 14, whichare incident on the objective 7, should have not only a component formoving the paraxial focal point but also a component for correctingspherical aberration due to the movement of the paraxial focal point.

[0082] Let us explain the spherical aberration correcting component withreference to FIG. 4. When rays with an inclination angle u are collectedat a point Q on the optical axis in the vicinity of the object-sidefocal point F of the objective 7, i.e. a distance ΔZ away from theobject-side focal point F, wavefront aberration W with respect to theobject-side focal point F is expressed by the following equation (5):

W=W _(F)+W_(SA)  (5)

[0083] where:

[0084] W_(F)=2ΔZ sin² (u/2)

[0085] W_(SA)=−2ΔZ sin⁴ (u/2)

[0086] In the above expression, W_(F) is wavefront aberration due to thefocal point shift, and W_(SA) is spherical aberration in terms ofwavefront aberration that occurs owing to the focal point shift. Thewavefront aberrations W_(F) and W_(SA) are derived from the Herschelcondition. That is, in order to obtain an image corrected for sphericalaberration at the plus-side shifted focal point 11 or the minus-sideshifted focal point 12, shown in FIGS. 2(a) and 2(b), a wavefrontconversion corresponding to the wavefront aberration W expressed by theabove equation (5) should be applied to the pre-correction illuminatinglight 15 by the wavefront converting element 5.

[0087] Next, a variation of the arrangement shown in FIG. 1 will bedescribed. In FIG. 1, two different elements are used as a laser lightsource and a light-detecting device, respectively. However, it is alsopossible to endow a single element with both the function of a lightsource and the function of a light-detecting device. That is, asemiconductor laser chip as used as a light source can also function asa photo-detector. Accordingly, if a semiconductor laser chip is used,the beam splitter 21 becomes unnecessary, and a laser feedbackmicroscope is constructed. If a gas laser is used as a light source,because it emits a narrow parallel beam, a beam expander should be usedin place of the collimation lens 4.

[0088] As the wavefront converting element 5, it is possible to use atransmission type wavefront converting element using a liquid crystalcell or the like. Alternatively, a reflection type wavefront convertingelement such as a membrane mirror may be used. Further, although in theforegoing arrangement the pre-correction illuminating light 15 enteringthe wavefront converting element 5 is a parallel beam, it may be adivergent beam or a convergent beam.

[0089] Further, the light-detecting optical path need not always bearranged to overlap the illuminating optical path. The light-detectingoptical path may be provided at the back of the sample 9 to detecttransmitted light. Alternatively, the light-detecting optical path maybe disposed at a side of the sample 9 to detect scattered light. Inparticular, when a pulse laser is used as the light source 6 to observethe sample 9 with fluorescence produced by two-photon excitation, it ispossible to obtain satisfactory resolution in the optical axis directioneven with a non-confocal optical system owing to non-linearcharacteristics inherent in two-photon fluorescence. In such a case, itis desirable to position the photo-detector closer to the sample thanthe illuminating optical path with a view to improving the lightdetection efficiency as well.

[0090] The second scanning optical microscope according to the presentinvention will be described below with reference to FIG. 5. The factthat the wavefront is kept constant during XY-scan will be explainedwith regard to a case where the pupil and the wavefront convertingelement are in conjugate positional relation to each other and alsoregarding a case where the pupil is not in conjugate relation to thewavefront converting element.

[0091] First, the influence of the scanning of the objective 7 on theimage will be described. The objective 7 is scanned along theXY-directions by the objective actuator 8. The amount of wavefrontconversion applied by the wavefront converting element 5 during thescanning is as expressed by the above-described equation (5). In thescanning optical microscope according to the present invention, theamount of wavefront conversion applied by the wavefront convertingelement 5 is kept constant independently of the values of X and Y.

[0092] Because the objective 7 is of the infinity corrected type, if thepost-correction illuminating light 17 is a plane wave as shown in FIG.3, the image-forming characteristics will not degrade even when theobjective 7 is scanned along the XY-directions. However, if scanningalong the XY-directions is performed after the focal point movement hasbeen made as shown in FIG. 2(a) or 2(b), the image-formingcharacteristics degrade. FIG. 5 shows a state where X-direction scanninghas been performed by ΔX in FIG. 2(b). In the figure, reference numeral39 denotes an ideal wavefront. Reference numeral 14 denotes theabove-described convergent beam wavefront. When ΔX=0, the convergentbeam wavefront 14 and the ideal wavefront 39 are coincident with eachother. When ΔX≠, however, a wavefront displacement shown by ΔW in thefigure occurs. The wavefront displacement is nothing but the wavefrontaberration with respect to the point Q where light is collected. Thewavefront aberration ΔW has an influence upon image-forming performancebut gives rise to no problem in practical application as long as it iswithin a predetermined range, i.e. provided that the Strehl ratio is 70%or more.

[0093] Tables 1 to 3 below show the results of simulation performed toexamine changes in the image-forming characteristics under variousconditions when the focal point movement and the scanning of theobjective along the XY-directions were performed simultaneously asstated above.

[0094] Table 1 shows the results of simulation in which the system wasarranged so that the wavefront converting element and the pupil of theobjective were conjugate to each other, and an optimum wavefrontconversion W for obtaining ΔZ=0.05 (mm) was applied. Scan range ΔXwithin which the Strehl ratio was 70% or more was obtained for thefollowing wavelengths of light and NA values:830 nm, 546.7 nm, and 248nm; and NA 0.5 to 0.9. It should be noted that the objective used was anideal objective having a focal length f_(OB)=3 (mm).

[0095] Similarly, Table 2 shows the results of simulation in whichf_(OB)=10 (mm), and ΔZ=0.05 (mm), and Table 3 shows the results ofsimulation in which f_(OB)=20 (mm), ΔZ=0.15 (mm), and NA was 0.5 to 0.7.TABLE 1 [f_(OB) = 3 (mm), ΔZ = 0.05 (mm), pupil conjugate] Wavelength(nm) NA ΔX (STR = 70%) 830 0.5 0.432 0.6 0.255 0.7 0.144 0.8 0.080 0.90.040 546.07 0.5 0.314 0.6 0.176 0.7 0.099 0.8 0.051 0.9 0.026 248 0.50.158 0.6 0.083 0.7 0.043 0.8 0.023 0.9 0.011

[0096] TABLE 2 [f_(OB) = 10 (mm), ΔZ = 0.05 (mm), pupil conjugate]Wavelength (nm) NA ΔX (STR = 70%) 830 0.5 1.348 0.6 0.815 0.7 0.451 0.80.243 0.9 0.113 546.07 0.5 0.988 0.6 0.558 0.7 0.300 0.8 0.151 0.9 0.073248 0.5 0.507 0.6 0.258 0.7 0.131 0.8 0.069 0.9 0.033

[0097] TABLE 3 [f_(OB) = 20 (mm), ΔZ = 0.15 (mm), pupil conjugate]Wavelength (nm) NA ΔX (STR = 70%) 830 0.5 1.113 0.6 0.587 0.7 0.309546.07 0.5 0.737 0.6 0.393 0.7 0.200 248 0.5 0.346 0.6 0.172 0.7 0.091

[0098] Let us take notice of the results for the wavelength 546.07 nmand NA 0.7, for example, in Tables 1 to 3. In Table 1, ΔX=0.099 (mm) .In Table 2, ΔX=0.300 (mm). In Table 3, ΔX=0.200 (mm). It can be saidthat the wavefront aberration ΔW is sufficiently small in the above scanrange ΔX. Therefore, if XY-scanning is performed within these ranges,the wavefront need not be changed in accordance with the scanning. Inother words, favorable image-forming performance can be obtained withthe wavefront kept constant. These hold true of the other wavelengthsand NA values.

[0099] It should be noted that, in FIG. 5, OA₂ is located on theright-hand side of OA_(1.) However, if the absolute value of ΔX is thesame, the image-forming characteristics of the optical system are thesame regardless of whether OA₂ is on the right-hand side of OA₁ or onthe left-hand side thereof. The same shall apply in the followingoptical systems, unless otherwise specified.

[0100] The above Tables 1 to 3 show the results of simulation in whichthe system was arranged so that the pupil of the objective and thewavefront converting element were conjugate to each other. The followingis a description of a case where the objective pupil and the wavefrontconverting element are not in conjugate positional relation to eachother.

[0101] Table 4 below shows the way in which image-formingcharacteristics change as the conjugate relationship between theobjective pupil and the wavefront converting element is graduallydestroyed. The objective used was an ideal objective having a focallength f_(OB)=10 (mm) and NA 0.7. Strehl ratios for ΔX=0 to 0.3 (mm)when the distance between the objective pupil and the wavefrontconverting element was changed from 0 to 300 mm were obtained(wavelength:546.07 nm). TABLE 4 [f_(OB) = 10 (mm), NA = 0.7, wavelength546.07 nm] Distance between objective and wavefront ΔZ converting ΔX(mm) (mm) Pupil element (mm) 0 0.1 0.2 0.3 0.05 Conjugate 0 (STR=) 10.96 0.86 0.701 Non- 10 1 0.96 0.96 0.701 conjugate 30 1 0.96 0.86 0.701100 1 0.96 0.865 0.702 125 0.991 0.953 0.86 0.701 150 0.979 0.94 0.8410.681 200 0.815 0.792 0.75 0.632 −0.05 Conjugate 0 1 0.956 0.845 0.671Non- 10 1 0.955 0.842 0.668 conjugate 30 1 0.955 0.842 0.667 100 1 0.9550.842 0.667 125 1 0.955 0.842 0.667 150 1 0.955 0.839 0.667 200 1 0.9550.84 0.658 300 1 0.955 0.838 0.658

[0102] The results shown in Table 4 reveal that the system of thepresent invention suffers minimum off-axis image degradation even whenthe pupil of the objective and the wavefront converting element are notin conjugate positional relation to each other. That is, when the scanrange is ΔX=0.2 (mm), for example, even if the pupil is 200 mm away fromthe wavefront converting element, a Strehl ratio of 0.75 or more can beobtained.

[0103] As has been stated above, the system of the present invention ischaracterized in that when the objective is scanned along a directionperpendicular to the optical axis, i.e. the X-direction, the wavefrontconversion applied by the wavefront converting element is kept constant.It is, however, a matter of course that the system of the presentinvention is also applicable to two-dimensional scan in the X-Y plane.

[0104] Regarding the third scanning optical microscope according to thepresent invention, the scan range of the objective will be describedbelow with reference to FIGS. 6 and 7.

[0105] As has been stated above, the XY-scanning of the objective has aninfluence on the image-forming characteristics. However, as long as itis within a predetermined range, the influence does not matter inpractical application, and satisfactory image-forming performance isobtained. That is, although it depends on various conditions, the scanrange AX where the Strehl ratio is 0.7 or more can be obtained accordingto the following expression:

ΔX≦0.66f_(OB)·λ/ (ΔZ·NA⁴)  (1)

[0106] where:

[0107] f_(OB):the focal length of the objective;

[0108] ΔZ:the amount of focal point movement caused by the wavefrontconverting element;

[0109] λ:the wavelength of the illuminating light;

[0110] NA:the numerical aperture of the objective.

[0111] The results shown in Tables 1 and 2 above are shown in FIGS. 6and 7, respectively, together with the curves of the above formula (1).It will be understood from FIGS. 6 and 7 that the curves of formula (1)agree with the results shown in Tables 1 and 2. That is, in the presentinvention, the scan range ΔX of the objective is controlled so as tosatisfy the condition (1). Thus, favorable image-forming performance canbe obtained with the wavefront kept constant.

[0112] The fourth and fifth scanning optical microscopes according tothe present invention will be described below with regard to areflecting mirror with an aperture.

[0113] Regarding the fourth scanning optical microscope according to thepresent invention, a basic arrangement for minimizing the loss of light,together with a variation thereof, will be described below withreference to FIGS. 8 and 9.

[0114] An LSM in which focal point movement is made by a wavefrontconverting element, and the loss of light is extremely small and hence abright image can be obtained can be realized by the arrangement shown inFIG. 8. In the illustrated arrangement, a laser light source 6 emitsilluminating light 2. The illuminating light 2 is magnified through abeam expander 20. The magnified illuminating light 2 passes through abeam splitter 21 and is collected through a convex lens 22. Thecollected light is incident on a reflecting mirror 42 having areflecting surface 24 and an aperture 25 (the reflecting mirror 42 willhereinafter be referred to as “apertured reflecting mirror”). Theincident light passes through the aperture 25 and is incident on areflection type wavefront converting element 26. The illuminating lightis subjected to wavefront conversion when reflected by the reflectiontype wavefront converting element 26. The wavefront-convertedilluminating light is reflected by the reflecting surface 24 and formedinto an approximately parallel beam through a collimation lens 27. Then,the illuminating light is collected on a sample 9 through an objective7. Viewing light from the sample 9 travels along a path reverse to theabove and is reflected by the beam splitter 21 and collected on aphoto-detector 29 through a convex lens 28.

[0115] The scanning optical microscope shown in FIG. 8 is similar to thearrangement shown in FIG. 1 in that the movement of the focal point andthe correction of spherical aberration due to the focal point movementare made by the reflection type wavefront converting element 26, and theXY-scanning of the objective 7 is performed by the objective actuator 8.

[0116] The fourth scanning optical microscope uses the aperturedreflecting mirror 42 as a device for leading illuminating light to thereflection type wavefront converting element 26. Accordingly, unlike theprior art using a beam splitter as shown in FIGS. 27 and 28, thisscanning optical microscope can reduce the loss of light to an extremelysmall quantity. Further, because a narrowed beam passes through theaperture 25, a confocal effect can be obtained by appropriately settingthe size of the aperture 25. The confocal effect is particularly usefulfor observation of a fluorescent sample. To perform fluorescenceobservation, it is desirable to use a dichroic mirror having appropriatewavelength characteristics in place of the beam splitter 21.

[0117]FIG. 9 shows the arrangement of another embodiment of the scanningoptical microscope using an apertured reflecting mirror. In thisembodiment, an apertured reflecting prism 23 comprising two rectangularprism members and a reflecting surface is used as an aperturedreflecting mirror. The apertured reflecting prism 23 has a reflectingsurface 24 at a cemented surface between the two prism members. Anaperture 25 is provided in a part of the reflecting surface 24. Further,a semiconductor laser chip 1 is used as a light source also serving as aphoto-detector to construct a laser feedback microscope.

[0118] Illuminating light 2 is collected in the aperture 25 through onlya convex lens 30 without using a beam expander. The arrangement of therest of the system is the same as that shown in FIG. 8. The arrangementshown in FIG. 9 is similar to that shown in FIG. 8 in that the movementof the focal point and the correction of spherical aberration due to thefocal point movement are made by the reflection type wavefrontconverting element 26, and the XY-scanning of the objective 7 isperformed by the objective actuator 8, and also in terms of the actionand effect of the apertured reflecting prism 23 to reduce the loss oflight.

[0119] It should be noted that an apertured reflecting mirror is alsoapplicable to a beam scan type LSM, although it involves problems suchas a change in the objective pupil position due to switching betweenobjectives and an increase in the overall size of the system due to thepresence of a pupil relay optical system. The application of anapertured reflecting mirror to a beam scan type LSM will be shown below.

[0120]FIG. 10 shows the arrangement of another embodiment of thescanning optical microscope using an apertured reflecting mirror. Inthis embodiment, a galvanometer mirror 47 is used as a scanning device,and two pupil relay optical systems 46 are arranged to place theobjective pupil 45, the galvanometer mirror 47 and the reflection typewavefront converting element 26 in conjugate relation to each other. Theportion of this embodiment that is not illustrated in the figure is thesame as that of the arrangement shown in FIG. 8.

[0121] The apertured reflecting mirror 42 is extremely effective inreducing the loss of light when focal point movement is effected by areflection type wavefront converting element not only in the objectivescanning type LSM shown in FIGS. 8 and 9 but also in the beam scan typeLSM using a galvanometer mirror or the like, which is shown in FIG. 10.As a device for beam scan, a polygon mirror or an AOM (acoustic-opticalmodulator) may also be used besides a galvanometer mirror.

[0122] Next, the fifth scanning optical microscope according to thepresent invention will be described with reference to FIGS. 11 to 13.The arrangement of the fifth scanning optical microscope is the same asthat shown in FIGS. 8 and 9. The following is a description of theaperture 25.

[0123] How the apertured reflecting mirror shown in FIGS. 8 and 9 canefficiently lead illuminating light to the wavefront converting elementwill be described below with reference to FIGS. 11, 12(a) and 13 incombination with FIGS. 8 and 9.

[0124] In FIGS. 8 and 9, the illuminating light 2 is collected throughthe lens 22 or 30 when incident on the apertured reflecting mirror 42 orthe apertured reflecting prism 23. The apertured reflecting mirror 42 orthe apertured reflecting prism 23 is positioned so that the positionwhere the illuminating light 2 is collected and the aperture 25 arecoincident with each other. With this arrangement, the illuminatinglight 2 passes through the aperture 25. Therefore, there issubstantially no loss of light when the illuminating light 2 passesthrough the aperture 25. After passing through the aperture 25, theilluminating light 2 becomes a divergent beam. The divergent beam isreflected by the reflection type wavefront converting element 26 tobecome a post-correction illuminating light 17. After further diverging,the post-correction illuminating light 17 is reflected by the reflectingsurface 24. At this time, a part of the illuminating light passesthrough the aperture 25 instead of being reflected. Therefore, there isa loss of light at the aperture 25. This is shown in FIG. 11. The amountof light lost at the aperture 25 can be evaluated at a plane 40 parallelto the reflection type wavefront converting element 26. Let us assumethat the radius of an outline defined by the outer periphery of the beamof post-correction illuminating light 17 when intersecting the plane 40is r_(Minc), and the radius of an outline of the post-correctionilluminating light 17 passing through the aperture 25 that is defined onthe plane 40 when the post-correction illuminating light 17 intersectsthe plane 40 is r_(H). On this assumption, the amount of light in therange of the radius r_(H) is the loss of the post-correctionilluminating light 17. If it is assumed that illuminating light in therange extending from the radius r. to the beam radius r_(Minc) is all(100%) reflected by the reflecting surface 24, the reflectance η_(H) iscalculated as follows.

[0125] Assuming that the post-correction illuminating light 17 is aGaussian beam with a beam radius r_(Minc) as shown in FIG. 13, theintensity distribution I(r) thereof is expressed by

I(r)=I _(O)·exp(−2r²/r_(Minc) ²)  (6)

[0126] From equation (6), the integral E(r) of the intensity within theradius r is expressed by $\begin{matrix}\begin{matrix}{{E(r)} = \quad {2\pi {\int_{0}^{r}{{{I(r)} \cdot r}{r}}}}} \\{= \quad {0.5\quad {I_{0} \cdot r_{minc}^{2} \cdot \pi}\left\{ {1 - {\exp \left( {{- 2}{r^{2}/r_{minc}^{2}}} \right)}} \right\}}}\end{matrix} & (7)\end{matrix}$

[0127] As has been stated above, a part of the post-correctionilluminating light 17 that falls within the range of the radius r_(H) islost. Therefore, with respect to the total amount of light within therange of the beam radius r_(Minc), the reflectance η_(H), i.e.efficiency, is expressed on the basis of equation (7) as follows:$\begin{matrix}\begin{matrix}{\eta_{H} = \quad {\left\{ {{E\left( r_{minc} \right)} - {E\left( r_{H} \right)}} \right\}/{E\left( r_{minc} \right)}}} \\{= \quad {\left\{ {{\exp \left( {2 - {2{r_{H}^{2}/r_{minc}^{2}}}} \right)} - 1} \right\}/\left\{ {{\exp (2)} - 1} \right\}}}\end{matrix} & (8)\end{matrix}$

[0128] It will be understood from the above equation (8) that η_(H) isdetermined by the ratio of r_(H) to r_(Minc).

[0129] The relationship between (r_(H)/r_(Minc)) and η_(H) is shown inTable 5 below. TABLE 5 (reflectance at apertured reflecting mirror)r_(H)/r_(Minc) 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 η_(H) 0.9770990.911083 0.809487 0.683287 0.544946 0.406420 0.277536 0.165037 0.072356

[0130] As will be understood from Table 5, if (r_(H)/r_(Minc))≦0.5,η_(H)≧0.54. It will also be understood that as (r_(H)/r_(Minc))decreases, η_(H), increases, and when (r_(H)/r_(Minc))=0.1, ¢_(H)reaches 0.977. Thus, the reflectance is extremely high. That is, theloss of light is favorably small.

[0131] In the case of the prior art using a beam splitter, even when apolarization beam splitter, which produces a relatively small loss oflight, is used, if light from the light source is random polarized, atleast a half of the random polarized light is lost when it is convertedinto linearly polarized light. In contrast, the present invention canprovide η_(H)≧0.54 by arranging the optical system so that(r_(H)/r_(Minc))≦0.5 regardless of whether light is polarized ornon-polarized. Consequently, the illuminating light can be led to thewavefront converting element more efficiently than in the case of theprior art using a beam splitter.

[0132] Although in the foregoing the shape of the aperture 25 in FIG.12(a) has been described as a circle having a radius r_(H), the shape ofthe aperture for producing the above-described effect of minimizing theloss of light is not necessarily limited to the circular shape. Forexample, the aperture shape may be elliptical, polygonal, a star-shape,a slit-like shape, or an irregular shape. Even if the aperture has sucha shape, the illuminating light can be efficiently led to the wavefrontconverting element as long as the optical axis extends through theaperture. When the aperture shape is not circular, the minimum valuer_(Hmin) of the length from the reflecting mirror edge (the boundarybetween the reflecting surface and the aperture) to the optical axisshould be regarded as r_(H). This is shown in FIGS. 12(b) and 12(c).

[0133] For the reasons stated above, it is desirable that the opticalsystem including the apertured reflecting mirror should satisfy thefollowing condition (2):

r _(Hmin)/r_(Minc)≦0. 5   (2)

[0134] where:

[0135] r_(Hmin):the minimum value of the length from the optical axis tothe reflecting mirror edge;

[0136] r_(Minc):the radius of wavefront-converted illuminating lightincident on the apertured reflecting mirror.

[0137] It should be noted that the optical system satisfying thecondition (2) means the optical system extending from the light sourceto the objective.

[0138] It should be further noted that the aperture 25 in the presentinvention is in confocal relation to the focal position of the objective7, and it is therefore extremely easy to perform confocal microscopicobservation by detecting a confocal signal. In such a case, it isdesirable that the shape and size of the aperture 25 should be madesuitable for confocal observation. For example, the shape and size ofthe aperture 25 should preferably be selected in conformity to the Airydisk diameter of viewing light. When the aperture 25 is adapted forconfocal observation, the size of the aperture 25 becomes smallinevitably. Therefore, (r_(H)/r_(Minc)) also becomes extremely small.Hence, η≈1. Consequently, there is almost no loss of light.

[0139] The sixth and seventh scanning optical microscopes according tothe present invention will be described below.

[0140] Regarding the sixth scanning optical microscope, a variation ofthe basic arrangement for minimizing the loss of light will be describedbelow with reference to FIG. 14. Another LSM in which focal pointmovement is made by a wavefront converting element, and the loss oflight is extremely small and hence a bright image can be obtained can berealized by the arrangement shown in FIG. 14.

[0141] A laser light source 6 emits illuminating light 2. Theilluminating light 2 passes through a beam splitter 21. Then, theilluminating light 2 passes through a convex lens 30 to enter a prism 31having a reflecting surface 32. The illuminating light is collected onthe reflecting surface 32 and reflected therefrom. Then, theilluminating light, which is now a divergent beam, is incident on areflection type wavefront converting element 26 where it is subjected towavefront conversion. The illuminating light exiting the reflection typewavefront converting element 26 passes through the prism 31 having thereflecting surface 32. Then, the illuminating light is formed into anapproximately parallel beam through a collimation lens 27 and collectedon a sample 9 through an objective 7. Viewing light from the sample 9travels along a path reverse to the above and is reflected by the beamsplitter 21 and collected on a photo-detector 29. The arrangement shownin FIG. 14 is similar to those shown in FIGS. 1, 8 and 9 in that themovement of the focal point and the correction of spherical aberrationdue to the focal point movement are made by the reflection typewavefront converting element 26, and the XY-scanning of the objective 7is performed by the actuator 8.

[0142] In this system, an optical element having a reflecting surfacedisposed at the position where the convergent beam is collected, thatis, the prism 31 having a reflecting surface, is used as a device forleading Illuminating light to the reflection type wavefront convertingelement 26. Therefore, the loss of light is extremely small in contrastto the prior art systems shown in FIGS. 27 and 28, which use beamsplitters. Reflected light from a region in the sample 9 that isconjugate to the reflecting surface 32 reaches the photo-detector 29 asviewing light 3. Conversely, reflected light from anywhere other thanthe conjugate region does not reach the photo-detector 29. That is, thesystem exhibits actions similar to those of a confocal optical system.By appropriately setting the size of the reflecting surface 32, aconfocal effect can be obtained. The confocal effect is particularlyuseful for observation of a fluorescent sample. To perform fluorescenceobservation, it is desirable to use a dichroic mirror having appropriatewavelength characteristics in place of the beam splitter 21.

[0143] An optical element having a reflecting surface, e.g. theforegoing prism 31 having a reflecting surface, is extremely effectivein reducing the loss of light when focal point movement is effected bythe reflection type wavefront converting element 26 not only in theobjective scanning type LSM shown in FIG. 14 but also in a beam scantype LSM using a galvanometer mirror or the like.

[0144] Regarding the seventh scanning optical microscope according tothe present invention, the reflecting mirror configuration will bedescribed below with reference to FIGS. 15 and 16.

[0145] How the optical element with a reflecting surface shown in FIG.14 can efficiently lead illuminating light to the wavefront convertingelement will be described below with reference to FIGS. 13, 15 and 16 incombination with FIG. 14.

[0146] In FIG. 14, the illuminating light 2 is converged through thelens 30 when entering the prism 31 having a reflecting surface. Theconverged illuminating light 2 is reflected by the reflecting surface 32provided at the focal position of the lens 30. There is almost no lossof illuminating light 2 when reflected by the reflecting surface 32. Theilluminating light 2 reflected from the reflecting surface 32 becomes adivergent beam and is then incident on the reflection type wavefrontconverting element 26. The illuminating light 2 reflected from thereflection type wavefront converting element 26 becomes apost-correction illuminating light 17. After further diverging, thepost-correction illuminating light 17 passes through the prism 31 havinga reflecting surface. At this time, a part of the illuminating light isreflected from the reflecting surface 32 instead of passing through it.The reflected part of the illuminating light is the loss of light.

[0147] The way in which the loss of illuminating light is produced isshown in FIG. 15. The amount of light lost at the reflecting surface 32can be evaluated at a plane 41 parallel to the reflection type wavefrontconverting element 26. Let us assume that the radius of an outlinedefined by the outer periphery of the beam of post-correctionilluminating light 17 when intersecting the plane 41 is r_(Ainc), andthe radius of an outline of the post-correction illuminating light 17reflected from the reflecting surface 32 that is defined on the plane 41when the post-correction illuminating light 17 intersects the plane 41is r_(Ainc). On this assumption, the amount of light in the range of theradius r_(M) is the loss of the post-correction illuminating light 17.Let us further assume that illuminating light in the range extendingfrom the radius r_(M) to the beam radius r_(Ainc) passes completely(100%) through the prism 31 having a reflecting surface, and thepost-correction illuminating light 17 is a Gaussian beam with a beamradius r_(Ainc) as shown in FIG. 13. On this assumption, thetransmittance η_(M) is determined in the same way as in the case ofequation (8), which expresses the reflectance η_(H) of the aperturedreflecting mirror, shown in FIGS. 8 and 9. It is only necessary toreplace r_(H) and r_(Minc) in equation (8) with r_(M) and r_(Ainc),respectively, as follows: $\begin{matrix}\begin{matrix}{\eta_{M} = \quad {\left\{ {{E\left( r_{Ainc} \right)} - {E\left( r_{M} \right)}} \right\}/{E\left( r_{Ainc} \right)}}} \\{= \quad {\left\{ {{\exp \left( {2 - {2{r_{M}^{2}/r_{Ainc}^{2}}}} \right)} - 1} \right\}/\left\{ {{\exp (2)} - 1} \right\}}}\end{matrix} & (9)\end{matrix}$

[0148] The relationship between η_(M) on the one hand and r_(Ainc) andr_(M) on the other in equation (9) is, needless to say, similar to therelationship between η_(H) on the one hand and r_(Minc) and r_(H) on theother in equation (8). Therefore, the relationship between(r_(M)/r_(Ainc)) and η_(M) agrees with the relationship between(r_(H)/r_(Minc)) and η_(H) in Table 5.

[0149] Although in the foregoing the shape of the reflecting surface inFIG. 16 has been described as a circle having a radius r_(M), the shapeof the reflecting surface for producing the above-described effect ofminimizing the loss of light is not necessarily limited to the circularshape. For example, the shape of the reflecting surface may beelliptical, polygonal, a star-shape, a slit-like shape, or an irregularshape. Even if the reflecting surface has such a shape, the illuminatinglight can be efficiently led to the wavefront converting element as longas the optical axis extends within the reflecting surface. Further, thereflecting surface 32 does not always need to be formed on alight-transmitting member (a plane-parallel plate or a prism) . Forexample, the reflecting surface 32 may be formed from a small reflectingmember with a reflecting surface having a necessary area. In this case,the reflecting member may be supported by a support member. When theshape of the reflecting surface is not circular, the minimum valuer_(Mmin) of the length from the reflecting mirror edge to the opticalaxis should be regarded as r_(M).

[0150] For the reasons stated above, it is desirable that the opticalsystem including the reflecting surface should satisfy the followingcondition (3):

r _(Mmin)/r_(Ainc)≦0.5  (3)

[0151] where:

[0152] r_(Mmin):the minimum value of the length from the optical axis tothe reflecting mirror edge;

[0153] r_(Ainc):the radius of wavefront-converted illuminating light atthe position of the reflecting mirror.

[0154] Regarding the eighth scanning optical microscope according to thepresent invention, the minimization of the loss of light and the sizereduction will be explained below with reference to FIGS. 17 and 18.

[0155] Another LSM in which focal point movement is made by a wavefrontconverting element, and the loss of light is extremely small and hence abright image can be obtained can be realized by the arrangement shown inFIG. 17.

[0156] An optical element 43 is supported by a support member 34. Theoptical element 43 functions as both a light-emitting part and alight-receiving part. Illuminating light 2 emitted from the opticalelement 43 is incident on a reflection type wavefront converting element26 through a collimation lens 33. The illuminating light 2 is subjectedto wavefront conversion when reflected by the reflection type wavefrontconverting element 26. The reflected light passes through thecollimation lens 33 to become an approximately parallel beam and is thencollected on a sample 9 through an objective 7. Viewing light from thesample 9 travels along a path reverse to the above and is collected onthe optical element 43. The arrangement shown in FIG. 17 is similar tothose shown in FIGS. 1, 8, 9 and 14 in that the movement of the focalpoint and the correction of spherical aberration due to the focal pointmovement are made by the reflection type wavefront converting element26, and the XY-scanning of the objective 7 is performed by the actuator8.

[0157] In this system, the beam splitters used in the prior art shown inFIGS. 27 and 28 are eliminated by disposing the optical element 43,which serves as both a light-emitting part and a light-receiving part,in the optical path. In this case, the loss of light can be minimized byusing an optical element and a support member that block a minimum ofincident light as the optical element 43 and the support member 34. Forexample, if a semiconductor laser chip is used as the optical element43, because its outer diameter is small, the area with which the opticalelement 43 blocks light is small even when it is disposed in the opticalpath as in this embodiment. Further, if the semiconductor laser chip isendowed with both the function of a light source and the function of alight-detecting element, it is possible to construct a laser feedbackmicroscope. As the support member 34, a transparent substrate, e.g. aglass substrate, should preferably be used.

[0158]FIG. 18 shows another embodiment of the eighth scanning opticalmicroscope according to the present invention. In this embodiment, oneend of an optical fiber 36 is attached to the center of a collimationlens 35. The other end of the optical fiber 36 is connected to a laserlight source (not shown) and a photo-detector (not shown). Accordingly,the end of the optical fiber 36 attached to the collimation lens 35functions in the same way as the optical element 43 in FIG. 17. Becausethe optical fiber 36 can be formed with a small diameter, even if it isdisposed in the optical path as in this embodiment, the area with whichthe optical fiber 36 blocks the light path is small. Thus, the loss oflight can be minimized. The arrangement of the rest of this system isthe same as that shown in FIG. 17. That is, the movement of the focalpoint and the correction of spherical aberration due to the focal pointmovement are made by the reflection type wavefront converting element26, and the XY-scanning of the objective 7 is performed by the actuator8.

[0159] The ninth scanning optical microscope according to the presentinvention will be described below with reference to FIGS. 19 and 20 andTable 6 below. The ninth scanning optical microscope is arranged suchthat illuminating light is incident obliquely on a wavefront convertingelement, as a variation of the arrangement for minimizing the loss oflight.

[0160] An LSM in which focal point movement is made by a wavefrontconverting element, and there is no loss of light and hence a brightimage can be obtained can be realized by the arrangement shown in FIG.19. A laser light source 6 emits illuminating light 2. The illuminatinglight 2 is formed into a parallel beam through a collimation lens 4 andpasses through a beam splitter 21. Then, the illuminating light 2 isincident on a reflection type wavefront converting element 26 at anincident angle θ_(PR). The illuminating light 2 is subjected towavefront conversion when reflected by the reflection type wavefrontconverting element 26. The reflected illuminating light 2 is collectedon a sample 9 through an objective 7. Viewing light from the sample 9travels along a path reverse to the above and is reflected by the beamsplitter 21 and collected on a photo-detector 29 through a convex lens28.

[0161] It should be noted that the ninth scanning optical microscope issimilar to the foregoing laser scanning microscopes in that the movementof the focal point and the correction of spherical aberration due to thefocal point movement are made by the reflection type wavefrontconverting element 26, and the XY-scanning of the objective 7 isperformed by the actuator 8.

[0162] To perform fluorescence observation, it is desirable to use adichroic mirror having appropriate wavelength characteristics in placeof the beam splitter 21. In this case, fluorescent light can be detectedmore efficiently than in the case of the prior art shown FIG. 27. Inaddition, the illuminating light can be led to the sample moreefficiently than in the case of the prior art shown in FIG. 28.

[0163] The above-described arrangement in which a light beam is incidentobliquely on the reflection type wavefront converting element isextremely effective in minimizing the loss of light not only in theobjective scanning type LSM shown herein but also in a beam scan typeLSM using a galvanometer mirror or the like.

[0164]FIG. 20 shows another embodiment of the scanning opticalmicroscope in which illuminating light is incident obliquely on thereflection type wavefront converting element. In this embodiment, oneend of an optical fiber 36 is placed at a position away from the opticalaxis of an objective 7 by a distance a. The other end of the opticalfiber 36 is connected to a laser light source (not shown) and aphoto-detector (not shown) . Accordingly, the first-mentioned end of theoptical fiber 36 functions as both a light-emitting part and alight-receiving part. Illuminating light 2 emerging from the opticalfiber 36 is collimated through a convex lens 37 and incident on areflection type wavefront converting element 26 at an incident angleθ_(PR). The illuminating light 2 is subjected to wavefront conversionwhen reflected by the reflection type wavefront converting element 26.The reflected illuminating light 2 passes through the convex lens 37 andthen passes through a collimation lens 27 to become an approximatelyparallel beam. Then, the illuminating light 2 is collected on a sample 9through an objective 7. Viewing light from the sample 9 travels along apath reverse to the above and is collected into the end of the opticalfiber 36 and detected by the photo-detector (not shown). Thisarrangement is similar to the foregoing in that the movement of thefocal point and the correction of spherical aberration due to the focalpoint movement are made by the reflection type wavefront convertingelement 26, and the XY-scanning of the objective 7 is performed by theactuator 8.

[0165] With this arrangement, because the optical fiber 36 can be formedwith a small diameter, the distance a shown in the figure can beminimized. Further, as the collimation lens 37, a lens having a longfocal length f₃₇ can be used from the viewpoint of design. This meansthat it is possible to reduce the incident angle θ_(PR) with respect tothe reflection type wavefront converting element 26. Thus, thearrangement is excellent from the viewpoint of image-forming performance(described below).

[0166] Let us explain the relationship between the incident angle θ_(PR)and the image-forming performance. When the incident angle of light raysis 0°, the reflection type wavefront converting element can completelycorrect spherical aberration at the position where light is collected byperforming wavefront conversion to provide a rotationally symmetricconfiguration. However, when the incident angle is large, an off-axisaberration component is produced. Consequently, it becomes impossible tomake satisfactory aberration correction. As a result, the image qualitydegrades. However, the image quality degradation is so small that it isignorable as long as the incident angle is within a certain range.

[0167] Accordingly, a simulation was performed to obtain the upper limitvalue of the incident angle θ_(PR) at which the Strehl ratio was 0.7 ormore when focal point movement and spherical aberration correction weremade with a reflection type wavefront converting element disposed at atilt at a position conjugate to the pupil of an objective. The resultsof the simulation are shown in Tables 6 to 9 below. It should be notedthat the objective used was an ideal objective of the infinity correctedtype. TABLE 6 [obliquely incident angle θ (°), (STR = 70%), f_(OB) = 10(mm), ΔZ = 0.05 (mm), pupil conjugate] NA 0.3 0.5 0.7 Wavelength 830 2313 8.5 (nm) 546.07 18.5 10.5 7 248 11 7 4.5

[0168] TABLE 7 [obliquely incident angle θ (°), (STR = 70%), NA = 0.5,ΔZ = 0.05 (mm), wavelength 546.07 (nm), pupil conjugate] f_(OB) (mm) 310 20 θ 11 10.5 10.5

[0169] TABLE 8 [obliquely incident angle θ (°), (STR = 70%), NA = 0.5,f_(OB) = 10 (mm), wavelength 546.07 (nm), pupil conjugate] ΔZ (mm) 0.020.05 0.1 0.2 θ 16.5 10.5 7.5 5.5

[0170] TABLE 9 [obliquely incident angle θ (°), (STR = 70%), f_(OB) = 3(mm), ΔZ = 0.02 (mm), pupil conjugate] NA 0.3 0.5 0.7 Wavelength 830 3520.5 14 (nm) 546.07 29 17 11.5 248 20 11.5 7.5

[0171] It will be understood from the above that the upper limit ofθ_(PR) is a function of NA, wavelength and ΔZ and unrelated to the focallength f_(OB) of the objective. The upper limit of θ_(PR) is given bythe following formula (4):

θ _(PR)≦50·NA^(31 1{square root}(λ·ΔZ) ³¹ ¹)  (4)

[0172] where:

[0173] θ_(PR):the angle (°) of incidence of the principal ray on thewavefront converting element;

[0174] ΔZ:the amount of focal point movement;

[0175] λ:the wavelength of the illuminating light;

[0176] NA:the numerical aperture of the objective.

[0177] The results shown in Table 6 and 9 above, together with thecurves of the above formula (4), are shown in FIGS. 21 and 22,respectively.

[0178] It will be understood from FIGS. 21 and 22 that the curves offormula (4) agree with the results shown in Tables 6 and 9. That is, theincident angle θ_(PR) with respect to the reflection type wavefrontconverting element can be determined from formula (4).

[0179] Regarding the tenth scanning optical microscope according to thepresent invention, the scheme of minimizing the loss of light by using atoric surface will be described below with reference to FIGS. 23 to 26,together with Table 10 below.

[0180] Another LSM in which focal point movement is made by a wavefrontconverting element, and there is no loss of light and hence a brightimage can be obtained can be realized by the arrangement shown in FIG.23. It should be noted that the term “aspherical toric surface” as usedin the following description means a surface configuration which has twoplanes of symmetry perpendicularly intersecting each other and in whicha curve intersecting these planes of symmetry is expressed by anaspherical sectional configuration.

[0181] A laser light source 6 emits illuminating light 2. Theilluminating light 2 is formed into a parallel beam through acollimation lens 4 and passes through a beam splitter 21. Then, theilluminating light 2 is incident obliquely on a reflection typewavefront converting element 44 controllable into an aspherical toricsurface configuration (hereinafter referred to as “toric wavefrontconverting element 44”). The illuminating light 2 is subjected towavefront conversion when reflected by the toric wavefront convertingelement 44. The reflected illuminating light 2 is collected on a sample9 through an objective 7. Viewing light from the sample 9 travels alonga path reverse to the above and is reflected by the beam splitter 21 andcollected on a photo-detector 29 through a convex lens 28.

[0182] In this system, focal point movement and the correction ofspherical aberration due to the focal point movement are made by thetoric wavefront converting element 44.

[0183] Further, the XY-scanning of the objective 7 is performed by anactuator 8 in the same way as in the foregoing laser scanningmicroscopes.

[0184] To perform fluorescence observation, it is desirable to use adichroic mirror having appropriate wavelength characteristics in placeof the beam splitter 21.

[0185] The above-described arrangement in which a light beam is incidentobliquely on the toric wavefront converting element 44 is extremelyeffective in reducing the loss of light not only in the objectivescanning type LSM shown herein but also in a beam scan type LSM using agalvanometer mirror or the like.

[0186] Let us explain why an aspherical toric surface is used as thesurface configuration of the wavefront converting element.

[0187] When a reflection type wavefront converting element is used inthe arrangement shown in FIG. 23 to make focal point movement, it isdesirable to control the surface configuration of the wavefrontconverting element into a free-form surface configuration from theviewpoint of correcting aberrations completely. However, because afree-form surface has no symmetry in configuration, it is not easy torealize a necessary free-form surface configuration precisely with anaccuracy on the order of the wavelength of light. Incidentally, theinventor of this application analyzed the configuration of thereflection type wavefront converting element actually required in thearrangement shown in FIG. 23 and, as a result, found that the requiredconfiguration is certainly a free-form surface in the strict sense ofthe term, but it has high symmetry and is extremely close to anaspherical toric surface. This will be explained below with reference toFIGS. 24 to 26.

[0188]FIG. 24 shows a model of an objective scanning microscope having areflection type wavefront converting element 44. A parallel illuminatinglight beam having a beam diameter of 4.2 mm and a wavelength of 830 nmis incident on the reflection type wavefront converting element 44 at anincident angle of 45°. The reflection type wavefront converting element44 has an elliptical effective-diameter area with a major diameter of5.94 mm and a minor diameter of 4.20 mm. The illuminating light issubjected to wavefront conversion, and the optical path thereof is bentthrough 90°by the reflection type wavefront converting element 44. Thereflected illuminating light enters an objective 7 positioned away fromthe reflection type wavefront converting element 44 by 10 mm on theoptical axis. The objective 7 is an ideal objective having a focallength of 3 mm and NA of 0.7. The surface configuration of thereflection type wavefront converting element 44 is formed so that theposition where light is collected by the objective 7 shifts to aposition away from the object-side focal point F by ΔZ=0.04 (mm). Let usmake a comparison between a case where the surface configuration of thereflection type wavefront converting element 44 is a free-form surfaceand a case where it is an aspherical toric surface.

[0189]FIG. 25 is a contour map (units:μm) showing the reflecting surfaceconfiguration when the surface configuration of the reflection typewavefront converting element 44 is a free-form surface. FIG. 26 is acontour map showing a reflecting surface configuration obtained bysubtracting an aspherical toric surface configuration having planes ofsymmetry in the ξ—ζ and η—ζplanes from the above-described free-formsurface. It should be noted that in FIGS. 25 and 26 the elliptical areawith a major diameter of 5.6 mm and a minor diameter of 3.9 mm is shownby the bold line. Contour lines within the elliptical area are shown bythe thin lines. It is observed in FIG. 25 that the reflecting surfaceconfiguration has a slightly asymmetric component with respect to theξ—ζ plane (it is obvious that the reflecting surface configuration issymmetric with respect to the η—ζplane). The maximum displacement is 8μm.

[0190] On the other hand, the maximum displacement in FIG. 26 is +0.04μm on the plus side and −0.06 μm on the minus side. Either of thedisplacements is less than 1% of the maximum displacement of theabove-described free-form surface, i.e. 8 μm.

[0191] Let us show that the difference in configuration between theaspherical toric surface and the free-form surface gives rise to noproblem in practical application.

[0192] In FIG. 24, a stop 48 with an aperture diameter of 3 mm was addedto the objective 7 with the toric wavefront converting element 44 placedas illustrated in the figure, and the stop 48 and the objective 7 werescanned together as one unit along the XY-directions. With this setup,the Strehl ratio was obtained. The results of the experiment are shownin Tables 10(a) and 10(b) below. In the tables, AY denotes the scanrange in the Y-direction. Regarding the distribution of theimage-forming characteristics in the XY-directions, it is obvious thatthe distribution is asymmetric with respect to the X-axis but symmetricwith respect to the Y-axis. Therefore, in Table 10(a), the Strehl ratiowas obtained in the ΔX range of 0 to +0.5 (mm). In Table 10(b), theStrehl ratio was obtained in the AY range of −0.5 to +0.5 (mm). TABLE 10(a) [f_(OB) = 3 (mm), NA = 0.5, wavelength 830 (nm), ΔZ = 0.04 (mm), ΔY= 0 (mm)] ΔX (mm) STR 0 0.997 0.2 0.935 0.4 0.754 0.5 0.628

[0193] TABLE 10 (b) [f_(OB) = 3 (mm), NA = 0.5, wavelength 830 (nm), ΔZ= 0.04 (mm), ΔX = 0 (mm)] ΔY (mm) STR 0.5 0.585 0.4 0.715 0.2 0.912 00.997 −0.2 0.958 −0.4 0.793 −0.5 0.672

[0194] It will be understood from the above that the Strehl ratio is 0.7or more in the ΔX range of 10.4 (mm) and in the AY range of ±0.4 (mm),and satisfactory image-forming performance can be obtained even with anaspherical toric surface in these ranges.

[0195] Scanning optical microscopes, e.g. laser scanning microscopes(LSMs), using a wavefront converting element according to the presentinvention provide the following advantageous effects.

[0196] With the first scanning optical microscope, scanning along adirection perpendicular to the optical axis is performed by scanning theobjective. Therefore, even when focal point movement is performed by thewavefront converting element, degradation of off-axis image-formingperformance is minimized. Moreover, from the viewpoint of thearrangement of the system, the objective pupil need not be conjugate tothe wavefront converting element.

[0197] With the second scanning optical microscope, when the objectiveis scanned along a direction perpendicular to the optical axis, it isunnecessary to change the wavefront conversion applied to illuminatinglight by the wavefront converting element. Therefore, the drive controlof the wavefront converting element is facilitated.

[0198] The third scanning optical microscope makes it possible to obtainan LSM that performs focal point movement by a wavefront convertingelement and has favorable image-forming characteristics.

[0199] The fourth to seventh scanning optical microscopes make itpossible to obtain an LSM suffering a minimum loss of light despite theuse of a reflection type wavefront converting element.

[0200] The eighth scanning optical microscope makes it possible toobtain an LSM suffering a minimum loss of light and compact in sizedespite the use of a reflection type wavefront converting element.

[0201] The ninth and tenth scanning optical microscopes make it possibleto obtain an LSM free from loss of light despite the use of a reflectiontype wavefront converting element.

What we claim is:
 1. A scanning optical microscope comprising: a lightsource; a wavefront converting element for applying a desired wavefrontconversion to illuminating light emitted from said light source; anobjective for collecting wavefront-converted illuminating light emergingfrom said wavefront converting element onto a sample; a detector fordetecting signal light emitted from said sample; and an actuator forscanning said objective along a direction perpendicular to an opticalaxis.
 2. A scanning optical microscope according to claim 1, whereinwhen said actuator scans one section of the sample perpendicular to theoptical axis with said objective, said wavefront converting elementapplies a constant wavefront conversion to said illuminating light.
 3. Ascanning optical microscope according to claim 1 or 2, wherein when anamount of movement of said objective along a direction perpendicular tothe optical axis is denoted by AX, the following condition (1) issatisfied: ΔX≧0.66f_(OB)·λ/ (ΔZ·NA⁴)  (1) where: f_(OB):a focal lengthof the objective; ΔZ:an amount of focal point movement caused by thewavefront converting element; λ:a wavelength of the illuminating light;NA:a numerical aperture of the objective.
 4. A scanning opticalmicroscope comprising: a light source; an optical element having apositive power for converting illuminating light emitted from said lightsource into a convergent beam; a reflecting mirror with an aperture; areflection type wavefront converting element for applying a desiredwavefront conversion to said illuminating light; an objective forcollecting said wavefront-converted illuminating light onto a sample;and a detector for detecting signal light emitted from said sample.
 5. Ascanning optical microscope according to claim 4, wherein an opticalsystem including said reflecting mirror with an aperture satisfies thefollowing condition (2): r _(Hmin)/r_(Minc≦)0.5  (2) where: r_(Hmin):aminimum value of a length from an optical axis to a reflecting mirroredge; r_(Minc):a radius of wavefront-converted illuminating lightincident on the reflecting mirror with an aperture.
 6. A scanningoptical microscope comprising: a light source; an optical element havinga positive power for converting illuminating light emitted from saidlight source into a convergent beam; a reflecting mirror placed at aposition where said convergent beam is collected; a reflection typewavefront converting element for applying a desired wavefront conversionto said illuminating light; an objective for collecting saidwavefront-converted illuminating light onto a sample; and a detector fordetecting signal light emitted from said sample.
 7. A scanning opticalmicroscope according to claim 6, wherein an optical system includingsaid reflecting mirror satisfies the following condition (3): r_(Mmin)/r_(Ainc)≦0.5  (3) where: r_(Mmin):a minimum value of a lengthfrom an optical axis to a reflecting mirror edge; r_(Ainc):a radius ofwavefront-converted illuminating light at a position of the reflectingmirror.
 8. A scanning optical microscope comprising: a light source; areflection type wavefront converting element for applying a desiredwavefront conversion to illuminating light emitted from said lightsource; and an objective for collecting wavefront-converted illuminatinglight onto a sample; wherein said light source also serves as a detectorfor detecting signal light emitted from said sample.
 9. A scanningoptical microscope comprising: a light source; a reflection typewavefront converting element for applying a desired wavefront conversionto illuminating light emitted from said light source; an objective forcollecting wavefront-converted illuminating light emerging from saidwavefront converting element onto a sample; and a detector for detectingsignal light emitted from said sample; wherein said reflection typewavefront converting element is placed in an optical path so as tosatisfy the following condition (4): θ_(PR)≦50·NA⁻¹{square root}(λ·ΔZ⁻¹)where: θ_(PR):an angle (°) of incidence of a principal ray on saidwavefront converting element; ΔZ:an amount of focal point movement; λ:awavelength of said illuminating light; NA:a numerical aperture of saidobjective.
 10. A scanning optical microscope comprising: a light source;a reflection type wavefront converting element for applying a wavefrontconversion to illuminating light emitted from said light source; anobjective for collecting wavefront-converted illuminating light emergingfrom said wavefront converting element onto a sample; and a detector fordetecting signal light emitted from said sample; wherein a reflectingsurface of said reflection type wavefront converting element iscontrollable into an aspherical toric surface configuration.